【作者】 张军；

【导师】 高明；

【作者基本信息】 天津医科大学 ， 临床医学（专业学位）， 2019， 博士

【摘要】 目的:甲状腺癌(thyroid carcinoma,TC)是最常见的内分泌恶性肿瘤,在过去数十年间其发病率在全世界范围内逐年增加,特别是女性甲状腺癌。甲状腺乳头状癌(papillary thyroid carcinoma,PTC)是甲状腺癌最常见的组织病理学类型,占所有甲状腺恶性肿瘤的90%以上。探讨甲状腺乳头状癌的分子机制和遗传改变是非常必要的。蛋白激酶A(protein kinase A,PKA)是丝氨酸-苏氨酸蛋白激酶超家族中研究最为透彻的成员,参与多种细胞的生物学调控。环化腺核苷一磷酸(Cyclic Adenosine monophosphate,cAMP)在真核生物中的主要功能是激活cAMP依赖性蛋白激酶(PKA)。PKA涉及多种肿瘤的发生和发展,正常的甲状腺细胞增殖受cAMP水平的影响极大,cAMP及其主要的细胞内靶蛋白PKA构成了与多种细胞功能有关的通路,其中细胞增殖和分化的调节最为重要。此外,cAMP在神经元和多种内分泌起源细胞中发挥促有丝分裂作用,并且cAMP下游通路的改变与多种内分泌肿瘤的发生相关。因此探讨PKA在甲状腺乳头状癌中是否对细胞功能产生影响,并且研究PKA如何调控细胞生物学功能改变的具体机制具有重要意义。Hippo信号通路在组织稳态中发挥重要作用,其确保了器官以适当的大小发育。Yes相关蛋白(Yes-associated protein,YAP)转录共激活因子是Hippo信号通路的主要效应物,并且被Hippo通路激酶LATS1/2(Large tumor suppressor 1/2)磷酸化和灭活。最近已经表明,YAP活性受G蛋白偶联受体(G Protein-Coupled Receptors,GPCRs)信号传导的调节。Gas-偶联受体的激活通常导致cAMP的积累,cAMP是一种重要的第二信使,具有多种生理功能,包括细胞增殖和分化。尽管进行了广泛的研究,但cAMP如何调节细胞增殖和分化的精确分子机制尚不完全清楚。本研究我们探讨了PKA对甲状腺乳头状癌的生物学功能影响,并阐述了PKA是否通过Hippo信号通路影响了甲状腺乳头状癌的进展。方法:1.选取天津医科大学肿瘤医院行手术治疗的甲状腺乳头状癌患者132例,其中有癌旁正常组织的标本40例。应用免疫组化的方法对组织标本进行染色,检测p-PKA的蛋白表达水平,对免疫组化结果进行判定评分,分析p-PKA在甲状腺乳头状癌与癌旁正常组织中的表达差异;并探讨p-PKA表达水平与甲状腺乳头状癌临床病理特征的关系及临床意义。2.PKA对甲状腺乳头状癌细胞增殖、迁移、凋亡以及细胞周期的影响。选取甲状腺乳头状癌细胞系TPC-1以及KTC-1作为研究对象,进行CCK8(Cell Counting Kit-8)以及克隆形成实验探讨PKA抑制剂KT5720、H89以及激活剂FI(Forskolin&IBMX)对甲状腺乳头状癌细胞增殖的影响;划痕实验探讨PKA抑制剂KT5720、H89以及激活剂FI对甲状腺乳头状癌细胞迁移的影响;Transwell实验探讨PKA抑制剂KT5720、H89以及激活剂FI对甲状腺乳头状癌细胞侵袭的影响;流式细胞术探讨PKA抑制剂KT5720、H89以及激活剂FI对甲状腺乳头状癌细胞周期和凋亡的影响。3.PKA通过Hippo信号通路抑制甲状腺乳头状癌的进展。应用PKA激活剂FI以及DMSO分别处理KTC-1进行RNA-seq,对RNA-seq进行生物信息学分析;应用Realtime PCR的方法检测YAP1的下游靶基因abcb1、ankrd、cat、ctgf、cyr6、gpatch4、imnb2以及txn的表达情况,进一步验证RNA-seq的结果。对132例甲状腺乳头状癌患者石蜡标本进行p-PKA和YAP1两个指标进行免疫组化染色,探讨p-PKA影响YAP1在甲状腺癌中的定位;加入DMSO、PKA抑制剂以及激活剂FI分别处理TPC-1和KTC-1细胞,应用蛋白质免疫印迹(Western Blot,WB)的方法检测Hippo信号通路主要蛋白以及凋亡、迁移相关通路蛋白水平的变化;随后我们使用DMSO、PKA抑制剂H89、KT5720及激活剂FI分别处理TPC-1及KTC-1,应用免疫荧光的方法观察YAP1的包浆定位,从而进一步验证PKA与Hippo信号通路的关系;接下来应用小干扰RNA技术敲弱Hippo信号通路关键蛋白LATS1/2,再次使用FI改变PKA磷酸化水平后观察YAP1磷酸化水平,定位及相关功能的变化;进一步应用克隆形成以及Transwell实验进行细胞功能回复实验;最后应用免疫缺陷的小鼠作为研究对象,通过尾静脉注射甲状腺乳头状癌细胞进入小鼠体内,饲养3-4周,通过动物活体成像技术验证是否成功建立肺转移模型,通过腹腔注射的方法给予DMSO以及FI,记录0天,2天,4天,6天,8天,10天,12天各组小鼠的体重,并应用动物活体成像技术,通过荧光亮度评估两组转移瘤的大小变化,12天后处死老鼠,并对小鼠肺组织进行免疫组化染色。结果:1.免疫组化结果显示:p-PKA在癌旁正常组织中表达明显高于甲状腺乳头状癌组织;p-PKA在甲状腺乳头状癌中的高表达率明显低于在癌旁正常组织中的表达(c2=20.803,P<0.001);进一步将p-PKA表达水平与甲状腺乳头状癌临床病理特征进行统计学分析,结果显示:在甲状腺乳头状癌中p-PKA与淋巴结转移(χ2=8.707,P=0.003)以及TNM分期(χ2=3.986,P=0.046)相关,而与年龄,性别,肿瘤大小,多灶,以及T分期无相关性(P>0.05)。在多因素分析中,我们发现p-PKA表达(OR 0.302;95%CI 0.136-0.675;P=0.004)和肿瘤大小(OR3.530;95%CI 1.555-8.015;P=0.003)是甲状腺乳头状癌淋巴结转移的独立预后因素。2.CCK8以及克隆形成实验表明PKA抑制剂KT5720、H89能够增强甲状腺乳头状癌细胞的增殖能力,PKA激活剂FI抑制甲状腺乳头状癌细胞的增殖能力;划痕实验和Transwell实验验证了PKA抑制剂KT5720、H89能够促进甲状腺乳头状癌细胞的迁移和侵袭,PKA激活剂FI抑制甲状腺乳头状癌细胞的迁移和侵袭;流式细胞术结果表明PKA抑制剂KT5720、H89对甲状腺乳头状癌细胞的周期及凋亡影响不明显,PKA激活剂FI诱导了甲状腺乳头状癌细胞的凋亡,并可以诱导甲状腺乳头状癌细胞的G1期阻滞。3.RNA-seq结果显示:PKA与Hippo信号通路相关,其可能是通过Hippo信号通路影响了甲状腺乳头状癌的生物学功能。对Hippo信号通路下游靶点YAP1的下游靶基因进行Realtime PCR检测,大部分下游基因出现表达下调,这进一步验证了RNA-seq的结果。免疫组化染色发现p-PKA低表达时YAP1定位在细胞核,p-PKA高表达时YAP1定位在细胞浆。Western Blot和免疫荧光检测发现:PKA可影响Hippo信号通路相关蛋白的磷酸化水平,同时,抑制迁移相关蛋白的表达及增加凋亡相关蛋白的表达。克隆形成以及Transwell实验进行细胞功能回复实验,进一步验证了PKA通过Hippo信号通路抑制甲状腺乳头状癌细胞的进展。最后我们进行了动物实验,通过小鼠肺转移模型,发现使PKA激活后,肺转移瘤增长明显减慢,对照组小鼠体重明显减轻,差异有统计学意义。通过免疫组化染色发现,实验组中p-PKA、p-LATS、p-YAP1、p53表现为阳性表达,而Vimentin表现为阴性。上述实验证明了PKA通过Hippo信号通路抑制甲状腺乳头状癌细胞的进展。结论:1.p-PKA在癌旁正常组织中表达明显高于甲状腺乳头状癌组织;在甲状腺乳头状癌中p-PKA与淋巴结转移以及TNM分期相关;p-PKA表达和肿瘤大小是甲状腺乳头状癌淋巴结转移的独立预后因素。2.PKA影响甲状腺乳头状癌细胞增殖、迁移、侵袭、凋亡以及细胞周期等生物学功能。3.PKA通过Hippo信号通路调控甲状腺乳头状癌细胞生物学功能的变化,从而抑制甲状腺乳头状癌的进展。更多还原

【Abstract】 Purpose:Thyroid cancer is the most common endocrine malignancy,and its incidence has increased year by year in the world over the past few decades,especially in women with thyroid cancer.PTC is the most common histopathological type of thyroid cancer,accounting for more than 90% of all thyroid malignancies.It is necessary to investigate the molecular mechanisms and genetic changes of PTC.Protein kinase A(PKA)is the most well-studied member of the serine-threonine protein kinase superfamily and is involved in the biological regulation of various cells.The main function of cAMP in eukaryotes is to activate cAMP-dependent protein kinase.PKA is involved in the development and progression of a variety of tumors,where normal thyroid cell proliferation is greatly affected by cAMP levels.cAMP and its major intracellular target protein,PKA,constitute pathways involved in a variety of cellular functions,of which regulation of cell proliferation and differentiation is of paramount importance.In addition,cAMP plays a mitogenic role in neurons and several endocrine-derived cells,and changes in cAMP downstream pathways are associated with tumorigenesis in several endocrine tumors.Therefore,whether PKA affects cell function in PTC is investigated,and it is important to study how PKA regulates the specific functions of cell biology.The Hippo signaling pathway plays an important role in tissue homeostasis,which ensures that organs develop at an appropriate size.The Yes-associated protein(YAP)transcriptional coactivator is the major effector of the Hippo pathway and is phosphorylated and inactivated by the Hippo pathway kinase LATS1/2(Large tumor suppressor 1/2).It has recently been shown that YAP activity is regulated by G protein coupled receptor signaling.Activation of Gas-coupled receptors typically leads to accumulation of cAMP,an important second messenger with multiple physiological functions,including cell proliferation and differentiation.Despite extensive research,the precise molecular mechanisms by which cAMP regulates cell proliferation and differentiation are not fully understood.In this study,we investigated the biological effects of PKA on PTC,and whether PKA affects theprogression of PTC through the Hippo signaling pathway.Method:1.A total of 132 patients with PTC who underwent surgery at the Tianjin Medical University Cancer Hospital were selected,including 40 paracancerous normal tissues.The tissue samples were stained by ICH,the expression level of p-PKA was detected,and the ICH results were scored.We analyzed the difference of p-PKA expression in PTC and paracancerous normal tissues,and discussed the relationship between the expression level of p-PKA and the clinicopathological characteristics of PTC and its clinical significance.2.The effect of PKA on proliferation,migration,apoptosis and cell cycle of PTC cells.CCK8 and clone formation experiments were performed to investigate the proliferation of PTC cells by PKA inhibitors KT5720,H89 and activator FI.Effects of scratch test on the migration of PTC cells by PKA inhibitors KT5720,H89 and activator FI;Transwell assay to investigate the effects of PKA inhibitors KT5720,H89 and activator FI on the invasion of PTC cells;Flow cytometry was conducted to investigate the effects of PKA inhibitors KT5720,H89 and activator FI on the cycle and apoptosis of PTC cells.3.PKA inhibits the progression of PTC through the Hippo signaling pathway.KTC-1was treated with PKA activator FI and DMSO for RNA-seq,and the bioinformatics analysis of RNA-seq was conducted.Realtime PCR was applied to detect the expressions of downstream target genes of YAP1,such as abcb1,ankrd,cat,ctgf,cyr6,gpatch4,imnb2 and txn.ICH staining of p-PKA and YAP1 in 132 paraffin-embedded specimens of PTC was performed to investigate the role of p-PKA in thyroid cancer localization.DMSO,PKA inhibitor and activator were added to process TPC-1 and KTC-1 cells,respectively.The changes in the levels of major proteins in the Hippo signaling pathway as well as proteins related to apoptosis and migration were detected by WB;Subsequently,DMSO,PKA inhibitor H89,KT5720 and activator FI were used to treat TPC-1 and KTC-1 of PTC cells,respectively,and immunofluorescence was used to observe the cytoplasmic localization of YAP1,so as to further verify the relationship between PKA and Hippo signaling pathway;next,use siRNA technology to knock down the key protein LATS1/2 of Hippo signalingpathway,and use FI to change PKA phosphorylation again.The level of YAP1 phosphorylation,localization and related functional changes were observed after phagocytosis;further,clone formation and Transwell experiments were used to perform cell function recovery experiments.Finally,immunodeficient mice were used as subjects to inject PTC cells into the small vein through the tail vein.Mice were housed for 2-3 weeks.The animal model was successfully used to verify whether the lung metastasis model was successfully established.DMSO and FI were administered by intraperitoneal injection for 0 days,2 days,4 days,6 days,8 days,10 days.The body weight of each group of mice in 12 days,and the in vivo size imaging technique was used to evaluate the size changes of the two groups of tumors by fluorescence brightness.The mice were sacrificed 12 days later,and the lung tissues of the mice were ICHly stained.Result:1.The results of ICH showed that p-PKA was significantly higher in paracancerous normal tissues than PTC(c2=20.803,P<0.001);the high expression rate of p-PKA in PTC was significantly lower than that in adjacent tissues;further analysis of the expression of p-PKA and the clinicopathological features of PTC.The results showed that in PTC,p-PKA was correlated with lymph node metastasis(2=8.707,p =0.003)and TNM stage(2=3.986,p =0.046),but not with age,gender,tumor size,multiple foci,and T stage(p >0.05).In the multivariate analysis,we found that the expression of p-PKA(OR 0.302;95% CI 0.136-0.675;P=0.004)and tumor size(OR 3.530;95%CI 1.555-8.015;P=0.003)is an independent prognostic factor for lymph node metastasis of PTC.2.CCK8 and colony formation experiments showed that PKA inhibitors KT5720 and H89 can promote the proliferation of PTC cells,PKA activator FI inhibits the proliferation of PTC cells;scratch assay and Transwell assay validate PKA inhibitors KT5720 and H89 can promote the migration and invasion of PTC cells.PKA activator FI inhibits the migration and invasion of PTC cells.Flow cytometry results showed that PKA activator FI induced the apoptosis of PTC cells,and could induce the G1 block of PTC cells.3.RNA-seq results show that PKA is associated with the Hippo signaling pathway,which may affect the biological function of PTC through the Hippo signaling pathway.Realtime PCR detection of downstream target genes of YAP1,a downstream target of Hippo signaling pathway,showed down-regulated expression of most downstream genes,which further confirmed the results of RNA-seq.ICH staining showed that YAP1 was located in the nucleus when p-PKA expression was low,and YAP1 was located in the cytoplasm when p-PKA expression was high.WB and immunofluorescence detection showed that PKA could affect the phosphorylation level of related proteins in the Hippo signaling pathway,and inhibit the expression of migration-related proteins and increase the expression of apoptosis-related proteins.Cloning formation and Transwell experiments for functional recovery experiments further validated the progression of PKA inhibition of PTC cells via the Hippo signaling pathway.Finally,we carried out animal experiments.Through the mouse lung metastasis model,it was found that after PKA activation,the growth of lung metastases was significantly slowed down,and the weight of the control group was significantly reduced.By ICH staining,p-PKA,p-LATS,p-YAP1,and p53 were positively expressed in the experimental group,while Vimentin was negative.The above experiments demonstrate that PKA inhibits the progression of PTC cells through the Hippo signaling pathway.Conclusion:1.The expression of p-PKA in adjacent tissues was significantly higher than that in PTC tissues;p-PKA is associated with lymph node metastasis and TNM staging in PTC;p-PKA expression and tumor size were independent prognostic factors for lymph node metastasis in PTC.2.PKA affects the proliferation,migration,invasion,apoptosis,cell cycle and other biological functions of PTC cells.3.PKA regulates the changes in biological functions of PTC through Hippo signaling pathway,thereby inhibiting the progression of PTC.更多还原